Flaviviruses have a global impact due to their widespread distribution and ability to cause encephalitis in humans and economically important domesticated animals. Of the approximately seventy viruses in the genus, roughly half have been associated with human disease. Several members of this group, such as dengue virus (DENV) and West Nile virus (WNV), are considered emerging or re-emerging pathogens because the incidence with which they encounter humans and cause disease is increasing each year at an alarming rate. Globally, DENV has become the most significant source of arthropod-borne viral disease in humans. Approximately 2.5 billion people (40% of the world's population) live at risk for DENV exposure across the globe, resulting in more than 100 million cases of DENV related illnesses each year.
The genome of flaviviruses such as DENV is a positive-stranded RNA. In the presence of non-structural proteins encoded by the virus, the RNA can be replicated within the cytoplasm of a host cell. A nucleic acid molecule that codes for all the proteins necessary for its replication in a cell is termed a “replicon”. If RNA encoding the DENV replicon is transfected into cells, the replicon can replicate. RNA-based replicons of Kunjin virus that carry a reporter gene have been described (Khromykh, et al. (1998), J Virol, 72:5967-77, Khromykh, et al. (1997), J Virol, 71:1497-505, Varnayski, et al. (1999), Virology, 255:366-75, Westaway, et al. (2005)) (U.S. Pat. No. 6,893,866). Such replicons can be transfected into stable or inducible cell lines to produce reporter viruses (Harvey, et al. (2004), J Virol, 78:531-8). Subgenomic replicons of Dengue virus have also been described (Holden, et al. (2006), Virology, 344:439-52) (Pang, et al. (2003);U.S. Patent Publication No. 2004/0265338)). A plasmid carrying a DNA-based version of a replicon that could be transfected into a cell directly (rather than an RNA transcript from the DNA) has been described for West Nile virus (Pierson, et al. (2005), Virology, 334:28-40). Replication-competent clones of West Nile virus have also been described that carry a green fluorescent protein (GFP) reporter virus (Pierson, et al. (2005), Virology, 334:28-40).
Four different serotypes of DENV are transmitted to humans through the bite of Aedes aegypti and Aedes albopictus mosquitoes. Clinical manifestations of exposure to DENV vary significantly (for review see (Gibbons, et al. (2002), Bmj, 324:1563-6)). Common clinical manifestations of dengue fever (DF) include a febrile illness accompanied by retroorbital, muscle and joint pain. While primary exposure to DENV is not associated with significant mortality, a small percentage of exposed individuals experience a more severe disease course referred to as dengue hemorrhagic fever (DHF). DHF, which is fatal in up to 10% of affected individuals, is most common in individuals that are sequentially infected with multiple different serotypes of the virus. Of significant concern is the rapid increase in the number of DHF cases during the past twenty years, resulting in over 450,000 cases of DHF each year (Monath, et al. (1996), Fields Virology, 2:961-1034). The increasingly common spread of different dengue serotypes is expected to increase the frequency of DHF significantly.
Dengue viruses are small spherical virions composed of three viral structural proteins, a lipid envelope, and a copy of the RNA genome (Kuhn, et al. (2002), Cell, 108:717-25, Mukhopadhyay, et al. (2003), Science, 302:248, Zhang, et al. (2003), Embo J, 22:2604-13). The cell biology of DENV entry into cells is poorly understood. To date, a cellular receptor for DENV has not yet been identified, although recent evidence suggests a role for DC-SIGN and/or DC-SIGNR during attachment and entry into primary dendritic cells (Navarro-Sanchez, et al. (2003), EMBO Rep, 4:723-8, Tassaneetrithep, et al. (2003), J Exp Med, 197:823-9). The role of the receptor is to bind virus particles on the cell surface and deliver them into the mildly acidic endosomal compartments of the cell, where the envelope proteins of the virus mediate fusion in a pH-dependent fashion.
The positive sense RNA genome of DENV is approximately 11 kb in length and encodes a single polyprotein that is cleaved by cellular and viral proteases into ten smaller functional subunits: three structural and seven non-structural (NS) proteins (Khromykh, et al. (1999), J Virol, 73:10272-80, Khromykh, et al. (2000), J Virol, 74:3253-63, Rice (1996), Fields Virology, 2:931-959). The structural proteins of DEN, which include the capsid, pre-membrane (prM) and envelope (E) proteins, are synthesized at the amino-terminus of the polyprotein and are present in the mature virus particle. The seven non-structural proteins encode all the enzymatic functions required for replication of the DENV genomic RNA, including a RNA-dependent RNA polymerase (NS5) (Rice (1996), Fields Virology, 2:931-959). The sequence encoding the DENV polyprotein is flanked by two untranslated regions (UTRs) that are required for efficient translation and genomic RNA replication (Khromykh, et al. (2003), J Virol, 77:10623-9, Khromykh, et al. (2000), J Virol, 74:3253-63, Novak, et al. (1994), Genes Dev, 8:1726-37). DENV RNA replication occurs in the cytoplasm at specialized virus-induced membrane structures (Mackenzie, et al. (1999), J Virol, 73:9555-67, Mackenzie, et al. (1998), Virology, 245:203-15). Viral particle biogenesis and budding occurs at the endoplasmic reticulum, and viruses are released through the secretory pathway of the cell (Lorenz, et al. (2003), J Virol, 77:4370-82, Mackenzie, et al. (2001), J Virol, 75:10787-99).
The ability of enveloped viruses to enter permissive cells is conferred by envelope glycoproteins incorporated into the viral membrane. Class II envelope proteins, encoded by the alpha- and flaviviruses, describe those that contain an internal fusion loop, lie flat across the surface of the native virion as dimers, and do not appear to form coiled-coils while mediating lipid mixing and fusion (reviewed in (Heinz, et al. (2000), Adv Virus Res, 55:231-69)). Like other class II fusion systems, DENV entry and fusion involves two separate proteins. The E protein plays a central role in virus entry by virtue of its capacity to bind receptor and mediate fusion in a pH-dependent fashion. The primary role of the second protein, prM, involves protecting newly formed particles from irreversible premature inactivation as they transit through mildly acidic compartments in the secretory pathway (Zhang, et al. (2003), Embo J, 22:2604-13). Other functions of prM have been demonstrated including directing E protein folding and trafficking (Lorenz, et al. (2002), J Virol, 76:5480-91). Structural studies suggest that all class II fusion proteins share a common structural design.
DENV virions are small spherical particles (50 nM) comprised of a lipid envelope incorporating 180 E glycoproteins arranged in a herringbone configuration (Kuhn, et al. (2002), Cell, 108:717-25). The capsid, prM and E components assemble at the endoplasmic reticulum to form an immature particle that buds into the lumen of the ER. Cleavage of the prM protein by the furin protease during trafficking to the cell surface (to generate the M protein), activates the fusion potential of the E protein, allowing the conformational changes that mediate fusion to occur upon exposure to low pH (Elshuber, et al. (2003), J Gen Virol, 84:183-91). Interestingly, expression of prM-E alone is sufficient for the production and secretion of subviral particles (SVPs) that, despite being smaller than mature viruses, retain the ability to mediate fusion in a manner analogous to mature particles containing capsid (Corver, et al. (2000), Virology, 269:37-46, Ferlenghi, et al. (2001), Mol Cell, 7:593-602, Heinz, et al. (1995), Vaccine, 13:1636-42). The ability to form subviral particles in the absence of any other viral proteins suggests that the forces that drive the process of particle biogenesis and budding reside in prM-E. Mature Dengue virus particles are approximately 50 nM in diameter and contain multiple copies of the viral capsid and the viral genomic RNA. Smaller 30 nM particles composed of prM-E proteins, called subviral particles, are also produced during virus infection. While subviral particles do not contain RNA or capsid, the E proteins on these particles are able to mediate receptor binding and fusion.
A primary target for neutralizing antibodies in a flavivirus infected host is the E glycoprotein present on the surface of the virus particle (Monath, et al. (1996), Fields Virology, 2:961-1034). Additionally, antibodies generated against prM and nonstructural protein-1 (NS1) have also been observed. Several lines of evidence support a significant role for such antibodies during virus clearance and the establishment of immunity following vaccination. For example, passive transfer of antibodies has been shown to confer protection in experimental systems with several flaviviruses, including tick bourne encephalitis (TBE), yellow fever virus (YF), Japanese encephalitis virus (JEV), WNV, and Saint Louis encephalitis virus (SLE). Studies in murine and hamster systems of WNV infection have reached similar conclusions. Several vaccine approaches are being developed, including the use of inactivated virus particles, live attenuated viruses, non-infectious subviral particles, subunit, and nucleic acid vaccines (Pugachev, et al. (2003), Int J Parasitol, 33:567-82). In many of these studies, particularly those in humans, the development of neutralizing antibodies is employed as a correlate of immunity and a measure of efficacy.
The development of a vaccine for DENV has been a significant challenge and the focus of considerable effort (Monath, et al. (1996), Fields Virology, 2:961-1034). While antibodies play a significant role in DENV immunity, the presence of DENV antibodies has also been linked to a more severe clinical outcome due to the ability of antibodies to facilitate DENV infection under some circumstances. While natural infection with one serotype of DENV results in generation of humoral immunity that protects against subsequent challenge with a homotypic virus, protection against other serotypes is transient. In fact, sequential exposure to different serotypes of DENV increases the likelihood of developing DHF. Pioneering work by Halstead and colleagues suggest that the presence of antibodies raised against the first serotype of DENV significantly impacts the outcome of a second exposure by allowing antibody dependent enhancement (ADE) of infection and the activation of both complement and the cellular immune system (Halstead (1988), Science, 239:476-81, Halstead (1989), Rev Infect Dis, 11 Suppl 4:S830-9, Halstead, et al. (1970), Yale J Biol Med, 42:311-28, Halstead, et al. (1977), J Exp Med, 146:201-17, Kliks, et al. (1989), Am J Trop Med Hyg, 40:444-51, Mongkolsapaya, et al. (2003), Nat Med, 9:921-7). Together, ADE has been linked to an increase in viral burden, increased vascular permeability, and a more severe disease course. One implication of these studies is that great care must be taken in the design of a vaccine against DENV to avoid a strategy that confers protection to only one serotype. Protection against only a single DENV serotype would increase the likelihood of an individual's chance of developing DHF should they encounter a second serotype of DENV. A tetravalent vaccine that simultaneously protects against all four serotypes of DENV is needed. Thus, characterizing not only the magnitude, but also the breadth, persistence, and specificity of the humoral response in response to vaccination is an important component of evaluating candidate vaccines and understanding pathogenesis in naturally infected individuals.
The standard method for detecting neutralizing antibodies to DENV is the plaque reduction neutralization test (PRNT) (Monath, et al. (1996), Fields Virology, 2:961-1034, Russell, et al. (1967), J Immunol, 99:291-6). Using this approach, the ability of an antibody to bind virus and neutralize its infectivity is measured as a reduction in the number of plaques formed following infection and subsequent propagation in cell culture. The PRNT approach involves the use of live infectious virus, and requires about a week for plaque formation and analysis. The quantitative power of plaque assays is limited by the number of wells examined and the number of plaques counted by the investigator. The latter process is somewhat subjective when plaque size and morphology is variable. The ability of flaviviruses to form plaques in infected cell monolayers is cell type-, and virus strain-dependent. Thus, the PRNT approach does not allow for the neutralizing capacity of antibodies to be detected using strains that plaque poorly, or on all permissive cell types, excluding many that may be relevant in vivo.
There is a need for better methods and compositions for the generation of pharmaceuticals and vaccines against flaviviruses, such as Dengue. The present invention fulfills these needs as well as others.